The industrial production using microorganisms is carried out for wide-ranging kinds of useful substances such as amino acids, organic acids, nucleic acid related compounds, antibiotics, carbohydrates, lipids, and proteins as well as alcoholic beverages and foods (e.g., miso (fermented soy paste) and shoyu (soy sauce)). The use of these substances is spreading into the wide field ranging from foods, pharmaceuticals, and commodities (e.g., detergents and cosmetics) to various chemical raw materials.
Improving productivity is one of important challenges for the industrial production of useful substances using microorganisms, and as a procedure therefor there has been performed the breeding of producing microorganisms by genetic techniques such as mutation. A producing microorganism has, particularly recently, come to be more efficiently bred using a recombinant DNA technology or the like owing to the development in microbial genetics and biotechnology, and the development of host microorganisms for gene recombination is under way. By way of example, there has been developed a strain obtained by further improving a microbial strain recognized as safe and good as a host microorganism, such as the Bacillus subtilis strain Marburg No. 168.
However, a microorganism originally has a wide variety of gene groups for accommodating itself to environmental changes in nature. Thus, the use thereof has not necessarily been efficient in the industrial production of a protein or the like, which employs limited production media. In particular, the microorganism has many types of proteolytic enzymes for degrading proteins to utilize them as nitrogen and carbon sources. These enzymes degrade a desired protein or the like, which has constituted a great barrier to the production of a foreign protein or the like.
Attempts have been made from long time ago to delete genes of these proteolytic enzymes (protease, peptidase, etc.) to prevent the degradation of desired proteins produced. Particularly for Bacillus subtilis, there have been reported, for example, a strain containing deletion of a gene encoding the major extracellular alkaline protease AprE or the neutral protease NprE or deletion of both of these genes (see Non-Patent Documents 1, 2, and 3), strains containing deletion of some of total 8 types of genes for extracellular and cell wall-bound proteases and peptidases (see Table 1 to be described later), and a strain containing deletion of all of the 8 genes for the proteases and peptidases (see Non-Patent Document 4). However, it has been demonstrated by an analysis or like by the present inventors that a proteolytic enzyme activity was still observed in a culture solution even of the microbial strain containing deletion of these 8 types of proteolytic enzyme genes, indicating the induction of degradation of a desired protein. Thus, there has been a need for identification of a causative proteolytic enzyme and a gene thereof.
The Bacillus subtilis aprX gene has been presumptively reported to encode AprX, an intracellular serine protease (see Non-Patent Document 5). However, it has not been reported at all that the AprX is present in a medium to degrade useful enzymes or proteins during the secretory production of these enzymes or proteins to reduce the yield thereof.
[Patent Document 1] Japanese Patent No. 288909
[Patent Document 2] Japanese Patent No. 3210315
[Patent Document 3] JP-A-2001-527401
[Non-Patent Document 1] J. Bacteriol., 158: 411 (1984)
[Non-Patent Document 2] J. Bacteriol., 160: 15 (1984)
[Non-Patent Document 3] J. Bacteriol., 160: 442 (1984)
[Non-Patent Document 4] Appl. Environ. Microbiol., 68: 3261 (2002)
[Non-Patent Document 5] Microbiology, 145: 3121-3127 (1999)